| IntroductionGastric carcinoma is one of the most common malignant tumor in the world, and the most popular malignant tumor in China. It was demonstrated that the carcinogenesis of gastric carcinoma is a multistep process participated with polygene.Tumor uppressor gene(TSG) is one of negative regulation signals on cell proliferation, differentiation and apoptosis process, and one of hot spots in oncological study. Regulation system of cell cycle is essential to sustain normal cell cycles. Furthermore, disorders of regulation can be caused by the abnormal of key factors in this regulation system, and will result in the abnormal of cell proliferation, unable to differentiate and apoptosis Promptly and occurrence of tumor.It has been popular presumed that abnormal methylation is the third mechanism except absence and mutation which would result in inactivation of TSGs, and plays an important role in tumorigenesis and development. Methylation is a natural modification style of DNA. This basic group modification process is catalyzed by DNA methyltransferase(DNMT) which would add a methyl(CH3-) to DNA. Most of methylated cytosine in human and animals located in CpG islands, and CpG in human genome is only account for 10%. Among them, 70% to 80% are in methylation situation. The methylation process of gene is a suppressive procedure of genetic expression. What is more, both methylation of TSGs and promoters of gene are closely related with tumorigenesis.hMLH1 gene and Rassfla gene are closely related to tumorigenesis and suppress cell proliferation. Inactivation of them will help absoluteness proliferation of tumor cells and promote tumor formation. Many studies found that methylation in promoter region of P16 gene was related to gastric carcinoma, hepatic carcinoma, colorectal carcinoma, esophageal carcinoma and so on. It will be helpful to reveal the regulation of cell cycle and provide clues to elucidate process of cell proliferation, differentiation, and cancerization with the study on this aspect. Furthermore, it will probably provide theoretical and experimental evidences to clinical diagnosis, treatment and prognosis of tumors.Methylationspecific PCR(MSP) were used in this experiment to detect methy lation level of hMLH1 gene and Rassfla gene in primary focus and adjuvant non-cancerous tissue of gastric carcinoma that aim at the investigation of relationship between methylation of hMLH1 gene and Rassfla gene and pathobiological behavior of metastasis of gastric carcinoma.MethodsFourty cases of matched paraffin embedded tissues from resected primary focus, metastasis and adjuvant non-cancerous tissue of gastric carcinoma used in this experiment were collected from gastric carcinoma patients treated in gastric surgical department of Cancer Hospital of Liaoning Province from January 2006 to August 2006. Among them, thirty were male and ten were female.The male female ratio(M-F ratio) was 3 vs 1 and mean age was 61.5±11.65(29 to 78). Carcinomas were staged according to TNM stage system of UICC and typed under Japanese gastric carcinoma management protocol. Adjuvant non-cancerous tissues were non-cancerous epithet lium mucosae. Both kinds of tissues were confirmed by pathologic diagnosis and no preoperative chemotherapy and radiotherapy were performed on these patients. Methylation level on promoter region of hMLH1 gene and Rassfla gene were detected with MSP method. Chisquared test or Fisher's exact propability test was used in comparison of ratio between groups. Experimental results were analyzed with the use of statistical package of SPSS(version 10.0), and differences were considered statistically significant when p value less than 0.05 as size of statistical test wasα=0.05.Results1. Positive rate of methylation on that 5 cm from promoter region of hMLH1 gene was 2.5%(1/40),12.5%(5/40) in 3cm from promoter region andin orresponding non-cancerous tissues. 10%(4/40) in 1cm from promoter region; And in promoter region was 32.5 %(13/40) cases of gastric carcinoma. Methylation levels in that of 5,3,1cm from promoter region tissues cancerous tissues were lower than with statistical significant(P<0.01). 2. Positive rate of methylation on that 5 cm from promoter region of RASSF1A gene was 5 % (2 / 40), 7.5 % (3 / 40) in 3cm from promoter region andin corresponding non-cancerous tissues.22.5 % (9 / 40) in 1cm from promoter region;. And in promoter region was 42.5% (17/40) in 40 cases of gastric carcinoma. Methylation levels in that of 5,3,1cm from promoter region tissues cancerous tissues were lower than with statistical significant (P＜0.01).3. Positive rate of methylation on promoter region of hMLH1 gene was 32.5 % (13 / 40) in 40 cases of gastric carcinoma, 8.69%(2/23) in Precancerous and 0% (0/24) in corresponding non-cancerous tissues that 5cm from promoter region tissues. Methylation levels in cancerous tissues, metastasis and non-cancerous tissues that 5cm from promoter region were statistical significant (P＜0.01).4. Positive rate of methylation on promoter region of RASSF1A gene was 42.5% (17/40) in 40 cases of gastric carcinoma, 17.39% (4/23) in Precancerous and 0% (0/24) in corresponding non-cancerous tissues that 5cm from promoter region tissues. Methylation levels in cancerous tissues, metastasis non-cancerous tissues were statistical significant (P＜0.01) among these tissues.5. Fourty patients were divided into lower lymphatic metastasis group (0～7)and highter lymphatic metastasis group (＞7) according to the number of metastasis lymphatic. Methylation levels of hMLH and RASSF1A genes were higher in highter lymphatic metastasis group than that of lower lymphatic metastasis group.the difference between the two groups has statistic significant (P＜0.05).6. Fourty tumors were divided into two groups according infiltrating depth.. Both of positive rates of methylation of hMLH and RASSF1A genes were higher in deeper groups than another group with statistical significant (P＜0.05).7. There is no statistic significance was found among methylation positive rates on promoter region of hMLH1,RASSF1A gene among histological differentiation degree,general type,growth patterns,ages and sex (P＞0.05).Conclusion1. Methylations on promoter region of hMLH1,RASSF1A genes are probably important molecule events during cancerization of gastric carcinoma. 2. Methylation rate of hMLH1 gene and RASSF1A gene in corresponding non-cancerous tissues that 5cm from promoter region tissues,Precancerous and promoter region were statistical significant.3. Methylation level on promoter region of hMLH1 genes and RASSF1A gene correlate with biological behaviours of gastric carcinoma, such as infiltrating depth and the number of metastasis lymphatic, but no statistic significance was found among methylation positive rates on promoter region of hMLH1,RASSF1A gene among histologicaldifferentiation degree,general type,growth patterns,ages and sex.And these figure could be used as one of prognostic indicators.4. Methylations on promoter region of hMLH 1 and RASSF1A could be used as one of prognostic indicators. |
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